Copyright © 2007, European Society of Cardiology
Nitric oxide suppresses NADPH oxidase-dependent superoxide production by S-nitrosylation in human endothelial cells
aBernard O’Brien Institute of Microsurgery, University of Melbourne, Fitzroy, Victoria 3065, Australia
bDepartment of Pharmacology, Monash University, Clayton, Victoria 3800, Australia
cCentre for Vascular Health, Monash University, Clayton, Victoria 3800, Australia
dDepartments of Surgery and Pharmacology, University of Melbourne, Victoria 3010, Australia
* Corresponding author. Tissue Engineering, Bernard O'Brien Institute of Microsurgery, University of Melbourne, 42 Fitzroy Street, Fitzroy, Victoria 3065, Australia. Tel.: +61 3 9288 4047; fax: +61 3 9416 0926. dusting{at}unimelb.edu.au
Objective: Endothelial NADPH oxidase is a major source of superoxide in blood vessels and is implicated in the oxidative stress accompanying vascular diseases, including atherosclerosis. Here we investigate the regulation of NADPH oxidase activity by nitric oxide (NO).
Methods: Human cultured microvascular endothelial cells (HMEC-1) were treated with the NO donors, diethylenetriamine (DETA)-NONOate, S-nitroso-N-acetylpenicillamine (SNAP) or sodium nitroprusside (SNP) for 0.5–24 h. Superoxide production was measured by lucigenin chemiluminescence and dihydroethidium fluorescence, while NADPH oxidase subunit expression was measured via Western blotting. S-nitrosylation was assessed using the 2,3-diaminonapthalene (DAN) assay, and via immunoblotting with an anti-nitrosocysteine antibody.
Results: Specific siRNA reduced Nox2 and Nox4 protein expression and markedly decreased superoxide production in HMEC-1. DETA-NONOate (10–300 µmol/L) suppressed superoxide production in HMEC-1 in a concentration- and time-dependent manner, which was not entirely attributable to stoichiometric reaction with NO, for the effect was observed more than 6 h after removing DETA-NONOate from solution. Similarly, sustained attenuation of superoxide production was achieved with SNP (10–100 µmol/L) and SNAP (10–100 µmol/L). The suppressive effect of NO was not dependent on (1) the sGC/cGMP/PKG pathway, (2) peroxynitrite-formation, (3) reduced protein expression of NADPH oxidase subunits or (4) dissociation of NADPH oxidase subunits. Treatment with NO caused S-nitrosylation of the crucial organizer subunit p47phox, and de-nitrosylation with UV light restored superoxide production.
Conclusions: NO causes sustained suppression of NADPH oxidase-dependent superoxide production in human endothelial cells by S-nitrosylation of p47phox. These findings highlight a novel approach by which vascular oxidative stress might be suppressed by NO donors.
KEYWORDS Nitric oxide; Endothelium; NADPH oxidase; Oxygen radicals
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  H. E. Marshall, E. N. Potts, Z. T. Kelleher, J. S. Stamler, W. M. Foster, and R. L. Auten Protection from Lipopolysaccharide-induced Lung Injury by Augmentation of Airway S-Nitrosothiols Am. J. Respir. Crit. Care Med., July 1, 2009; 180(1): 11 - 18. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. J. Hong and J. L. Garvin Nitric oxide reduces flow-induced superoxide production via cGMP-dependent protein kinase in thick ascending limbs Am J Physiol Renal Physiol, May 1, 2009; 296(5): F1061 - F1066. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Girouard, G. Wang, E. F. Gallo, J. Anrather, P. Zhou, V. M. Pickel, and C. Iadecola NMDA Receptor Activation Increases Free Radical Production through Nitric Oxide and NOX2 J. Neurosci., February 25, 2009; 29(8): 2545 - 2552. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Saito, A. Uppal, G. Byfield, S. Budd, and M. E. Hartnett Activated NAD(P)H Oxidase from Supplemental Oxygen Induces Neovascularization Independent of VEGF in Retinopathy of Prematurity Model Invest. Ophthalmol. Vis. Sci., April 1, 2008; 49(4): 1591 - 1598. [Abstract] [Full Text] [PDF]
|
|